Author response:
eLife Assessment
In this valuable manuscript, the authors tackle a highly relevant question in biology: how cells integrate attractive and repulsive cues to achieve directed migration. They present solid data demonstrating that two wunen genes act as negative regulators of Hedgehog signalling, thereby enabling efficient primordial germ cell (PGC) migration in Drosophila embryos. Beyond its immediate scope, this work has broader implications, particularly for understanding key mechanisms underlying complex processes such as cancer metastasis, where the coordinated interpretation of guidance cues is critical.
Thank you for the reviews and the overall assessment of our manuscript. It is our impression that both the reviewers and the senior editor find the study interesting and potentially of general relevance. The reviewers have made specific suggestions to improve the manuscript. They have also recommended ways to uncover the mechanistic basis to add to the broad appeal of the findings.
To begin with, we would like to point out that since the discovery of Wunen in 1996 by Ken Howard and colleagues, a number of genetic and molecular studies have attempted to identify and characterize the putative target(s) of the two lipid phosphate phosphatase(s). We and others have shown that Hh acts as a guidance signal for the migrating PGCs. Our data demonstrating the ability of Wunen(s) to attenuate Hh signaling constitutes an important step in elucidating the molecular underpinnings of the repulsive activity of Wun(s) during PGC migration.
Thus, we feel the need to share these findings with the scientific community at this juncture. In the following, we will summarize our response to the relevant points included in the individual public critiques of the reviewers without going into specific details.
Public Reviews:
Reviewer #1 (Public review):
This manuscript addresses how PGCs migrate towards SGPs in the Drosophila embryo. It's been shown that Hh produced by SGPs acts as an attractive cue, and that Wunnen(s) act as repulsive cues. In this work, the authors propose that Wun and Wun2 refine PGC guidance by attenuating Hedgehog signalling coming from other tissues.
Overall, the study is potentially interesting and could make an important contribution to the field. The data shown support the idea that Wun/Wun2 negatively regulate Hh signalling and produce PGC migration phenotypes associated with Hh. However, in my opinion, there are two major questions that should be addressed.
(1) Which is the mechanism by which Wun/Wun2 attenuates Hh signalling? The authors propose that Wun/Wun2 block Hh ligand transmission, but their data could also be explained by other possibilities, such as altered Hh production, uptake, retention or degradation, among others. The authors should either show the effect of Wun/Wun2 in Hh transmission mechanistically or attenuate their claim.
(2) How do Wun/Wun2 attenuate Hh signalling in PGCs? The authors propose that Wun/Wun2 function both in somatic tissues and in PGCs, but these two sites of action may have very different mechanistic implications. In the soma, Wun/Wun2 could affect Hh transmission, but a PGC-autonomous role cannot be explained simply by reduced Hh ligand transmission from producing cells; it would more likely involve ligand uptake, receptor trafficking, intracellular degradation or altered PGC responsiveness. This distinction should be central to the interpretation of the data.
We thank the reviewer for recognizing the importance of the problem and we are sensitive to both the points of criticism regarding the mechanism(s) Wunen(s) may employ to downregulate Hh signalling.
The reviewer correctly pointed out that we singled out Hh transmission as the putative target of Wunen(s) which need not be the case. We agree with this assessment and would like to thank the reviewer for pointing us in the right direction(s). Indeed, Wunen(s) could act at several different levels to regulate Hh signalling including “Hh production, uptake, retention or degradation”. We will modify the text to incorporate these possibilities in the appropriate sections of the manuscript.
The only reason for the emphasis on the ‘Hh transmission’ in the text was to contrast it with Hmgcr which acts in a qualitatively opposite manner. Hmgcr potentiates Hh signalling by altering the range/strength of the Hh ligand in the embryonic context. This was also confirmed in the wing discs and adult wings as hmgcr mutants could dominantly suppress the wing duplications and abnormalities induced by the ‘gain of function’ allele of hh (hhMRT). Upon compromising hmgcr, Hh ligand was shown to be sequestered in the Hh producing cells in the ectoderm. However, we have not carried out similar experiments to either rule in or rule out the different possibilities suggested by the reviewer. We will ensure that the claims made in the manuscript will appropriately reflect the scope of the analysis and the related arguments will be suitably modified.
The reviewer also makes a very critical point regarding cell autonomous v/s cell non autonomous activities of Wun(s). We have briefly mentioned the possible role of individual Wun(s) in the SGPs/mesoderm as well as within the PGCs. It has not escaped our notice that Wun(s) could regulate Hh internalization within the PGCs or its subcellular compartmentalization (within the ER, golgi or lysosomes). Wunen(s) could also act at the level of Hh reception by changing the activity/localization of Hh receptors, either Smoothened or Patched and could influence the outcome of the signaling pathway in a multi-pronged manner.
We appreciate the thoughtful suggestions and as recommended, future analysis will focus on these aspects. In our view, data included in the present version of the manuscript are novel and sufficient to argue a functional relationship between Wun(s) and Hh signalling which is qualitatively antagonistic to Hmgcr.
Reviewer #2 (Public review):
Summary:
In this submission, Roy et al. examine the process of Drosophila PGC migration. Directed cell migration requires the concerted activities of chemoattractants and repellents to guide cells to the correct locale. In their submission, the authors describe a role for regulated Hedgehog (Hh) signaling to inform PGC migration. In prior work, the authors reported that Hmgcr potentiates Hh signaling, providing a permissive axis. A gap in the field, however, was the identification of the repulsive cues that guide PGCs out of the midgut and toward the future gonad. In the current work, the authors report that two wunen genes (wunen and wunen 2) inhibit Hh signaling, thereby repressing Hh activity. The model is that Hmgcr and wunen(s) balance the transmission of Hh signals to enable effective PGC migration.
Strengths:
A strength of this work is the comprehensive genetic analysis performed by the authors. The authors examine zygotic versus maternal contributions, autonomous versus non-autonomous requirements, and use a variety of RNAi and mutant allele combinations to examine genetic requirements and interactions. Another strength is that the data presented are generally clear and well quantified. Insets are provided to enhance visualization, and relevant data are quantified through replicated experiments.
Weaknesses:
Weaknesses of the work include a lack of biochemical data to validate some of the proposed interactions. Although the authors do report lipidomics data, little is done with these findings to validate or place the results in the context of a mechanistic model. Despite these issues, the conclusions stated are generally well supported by the results.
We would like to thank the reviewer for their positive feedback and a succinct description of the findings reported in the manuscript.
We agree that the mechanistic basis of DAG accumulation was not explored in this manuscript. Prior work in the Ratnaparkhi and Kamat labs identified a Serine hydrolase that functions as a phospholipase C in biochemical assays (Kumar et al., 2024, Biochemistry 63:3000-3010). We have since conducted several genetic experiments, and preliminary data indicate that, in the embryonic context, mutations in the specific Phospholipase C display phenotypes analogous to wun(s). We hope to present these data along with the comparative molecular and biochemical analysis in the near future.
